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Abstract

Nonribosomal Peptide Synthetases (NRPSs) are modular multidomain enzymes that produce a wide variety of peptide natural products. These natural products, found in both bacteria and fungi, include antibiotics, antifungals, anticancers, immunosuppressants, and other types a therapeutics. Each NRPS module consists of a set of core domains including: an adenylation domain for amino acid activation, a peptidyl carrier protein for substrate transport between catalytic domains, and a condensation domain for peptide bond formation. The nascent natural product is passed from module to module in an assembly- line fashion until a terminal module containing a thioesterase domain is reached, and the natural product is released. The modular nature of NRPSs makes them ideal candidates for combinatorial biosynthesis where novel natural products are produced by swapping individual domains in an NRPS system to alter the final product. In order to have regular success in the production of these altered natural products, a complete understanding of the NRPS system is required. Presented within is work geared towards an understanding of NRPSs both structurally and functionally. In chapter two the structures of two terminal module NRPSs in two distinct catalytic conformations is presented. This provides a look at catalytically relevant domain interactions during biosynthesis, and provides a new model for the NRPS natural product biosynthetic cycle. These structures, along with negative stain electron microscopy, suggest that a single NRPS module is more dynamic than previously considered. In chapter three the linker region between the adenylation domain and PCP is examined. In multidomain NRPSs this linker region contains a conserved motif that appears to play two important roles in natural product biosynthesis. First this linker anchors a conserved coil motif that contains a catalytic residue for amino acid activation. This anchoring allows for proper placement of this catalytic residue when the adenylation domain is in the amino acid activating conformation. The anchoring role of the linker motif may also assist in coordinating the movement of the PCP with the movement of the C- terminal subdomain of the adenylation domain. Chapter four examines the structure and function of MbtH-like proteins (MLPs). These small non-catalytic proteins are required by some adenylation domains for amino acid activation. The extent of their role in natural product biosynthesis and the exact mechanism is unclear. However, presented are two structures of the same NRPS module bound to two different MLPs, the native MLP YbdZ and a non-native MLP, PA2412 from Pseudomonas aeruginosa. These MLPs bind to the N-terminal subdomain of the adenylation domain in the same location and conformation. Other difference in the modular architecture of the structures provide further insights into NRPS natural product biosynthesis. Finally, in order to structurally examine the interaction between the upstream PCP and the condensation domain, an interaction that has yet to be characterized, mechanism based inhibitors were designed to trap this interaction. This chapter examines the effectiveness of theses inhibitors via surface plasmon resonance. Binding data supports the use of these inhibitors for structural characterization via crystallography and potentially cryo electron microscopy.